Talbot and White Plant Methods 2013, 9:36
http://www.plantmethods.com/content/9/1/36
METHODOLOGY
PLANT METHODS
Open Access
Methanol fixation of plant tissue for Scanning
Electron Microscopy improves preservation of
tissue morphology and dimensions
Mark J Talbot* and Rosemary G White
Abstract
Background: It is well known that preparation of biological (plant and animal) tissues for Scanning Electron
Microscopy (SEM) by chemical fixation and critical point drying results in shrinkage of tissues, often by up to 20-30%,
depending on the tissue type and fixation protocol used. We sought to identify a protocol that would preserve
tissue size and morphology better than standard chemical fixatives and dehydration regimes. We compared a
range of processing techniques by quantifying changes in tissue size and recording details of surface morphology
using leaf tissues from three commonly studied species; Arabidopsis thaliana, barley and cotton.
Results: All processing protocols altered tissue dimensions. Methanol fixation and dehydration, followed by a
further short (1 h) dehydration step in ethanol and critical point drying (which was based on a previously published
method), preserved tissue dimensions most consistently of all protocols tested, although it did cause 8% shrinkage
in all three species. This protocol was also best for preservation of surface morphology in all three species. We
outline a recommended protocol and advise that the method is best trialled for different tissues, especially thicker
or larger samples.
Conclusions: This study shows that simultaneous fixation and dehydration in methanol followed by ethanol results
in better preservation of dimensions and morphology of critical point dried plant tissues than other fixation and
dehydration procedures. It is a quick and simple method, and requires standard SEM preparation equipment.
Keywords: Critical point drying, Plant cell morphology, Arabidopsis thaliana, Hordeum vulgare, Gossypium hirsutum
Background
A well-known artefact of preparing biological tissue for
Scanning Electron Microscopy (SEM) is the shrinkage of
tissue during fixation, dehydration and critical point drying (CPD) steps. In the past, these artefacts were documented largely by comparing the surface morphology of
animal and some plant tissues prepared using different
protocols. Several studies showed that after ‘conventional’ fixation with glutaraldehyde, dehydration with
ethanol followed by CPD, plant and animal tissues can
shrink to approximately 67-75% of their original size,
measured as either volume, length, or width [1-3]. The
total change in size arises from cumulative shrinkage in
* Correspondence: [email protected]
Commonwealth Scientific and Industrial Research Organisation, Division of
Plant Industry, Canberra, ACT 2601, Australia
each processing step, particularly the dehydration step
[2,4], and varies with different specimens [1].
Earlier investigations into the causes of tissue shrinkage found that chemical fixation was not critical for
good preservation [2,4]. In fact, Boyde and Boyde (1980)
showed that unfixed potato tuber tissue processed
through dehydration and CPD shrank less (15%) than
glutaraldehyde fixed tissue (30%; [2]). Another source of
shrinkage was the critical point drying process and postCPD storage, which could be due to retention of water
or ethanol in the tissue after drying, which then slowly
evaporated during storage [2,5].
Given that omission of aqueous fixation might improve
preservation of plant tissue [2] we sought to identify a
routine procedure that would maintain tissue size and
morphology for SEM examination. Although cryo-fixation
(plunging into liquid nitrogen or propane) has been
shown to be optimal for preservation of morphology [6], it
© 2013 Talbot and White; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Talbot and White Plant Methods 2013, 9:36
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requires dedicated, expensive equipment and results in an
approximately 9% increase in tissue volume due to ice
crystal formation during freezing [6,7]. Also, once prepared, the tissue cannot be stored for future imaging.
Freeze-drying is superior to CPD for preserving animal
tissue dimensions (e.g., [5]), but for plant [8,9] and fungal
[7] tissues it gives results similar to glutaraldehydefixation and CPD-processing at best, presumably because
these tissues contain considerably more water.
Fixation and dehydration of plant tissue with methanol
has been reported to improve preservation compared to
conventional techniques [10,11]. This method has been
used to document tissue morphology (e.g., [12-14]) and
for epidermal cell size analysis [15] by SEM, and also to
preserve roots for widefield light microscope imaging
[16]. Methanol processing was shown to have little or no
effect on coleoptile dimensions, whereas conventional
formaldehyde-acetic acid-alcohol (FAA) or glutaraldehyde fixation caused tissue shrinkage [10]. However, the
effect of methanol processing on dimensions of critical
point dried tissue for SEM has not been investigated.
This is important when using SEM for quantitative analysis, for example, of cell size [15].
This study aimed to identify an SEM preparation
method that preserves close to original tissue morphology and dimensions. A protocol based on solvent
fixation is an attractive alternative to conventional
SEM fixatives since tissue is simultaneously fixed and
dehydrated [10] and can then be transferred directly
to the CPD. We trialed methanol and other organic
solvents (ethanol and acetone) as alternatives to conventional SEM fixatives and quantified changes in tissue dimensions after processing through CPD. We
provide a detailed comparison of conventional fixation
methods with solvent-based protocols and conclude
that the methanol-ethanol protocol is generally the
best for preservation of plant tissues for SEM.
Results
To identify a robust SEM preparation method that
caused the least modifications to tissue dimensions and
morphology we compared the effects of seven different
fixation protocols on the preservation of leaf samples
from Arabidopsis thaliana, barley and cotton. The first
two fixatives, FAA and 3% glutaraldehyde, are routinely
used for SEM. Published methods using these fixatives
were followed with little or no modification to provide a
basis for comparison with the five solvent-based fixation
procedures we tested. Since 70% ethanol is a quick, relatively non-toxic fixative used for a variety of purposes,
including fixation of samples in the field, we included
this protocol. We were particularly interested in testing
the methanol-based protocols recommended from previous work [10]. Methanol fixation was followed by either
Page 2 of 7
further dehydration in methanol and critical point drying in methanol (‘Methanol’), or by ethanol dehydration
and critical point drying in ethanol (‘Methanol-ethanol’).
We also investigated 100% ethanol and acetone as fixatives since these solvents are commonly used to dehydrate
tissues for SEM and Transmission Electron Microscopy
(TEM). Treatment effects were monitored by expressing
the final area of processed leaves or leaf pieces as a percentage of the area of unfixed material, and by imaging
leaf morphology.
Effect of fixation methods on tissue area
Cotton leaves generally showed the least change in
area after any fixation protocol, followed by barley and
A. thaliana (Figure 1). For each species, a different protocol gave optimal results, for example, 70% ethanol fixation was best for cotton, and 100% ethanol best for
barley. However, although 100% ethanol and acetone
appeared preferable to the methanol-ethanol fixation
for A. thaliana, the latter protocol was most reliable,
indicated by the low standard errors in Figure 1. The raw
data show that replicate samples fixed in 100% ethanol or
acetone could either swell or shrink (Additional file 1 and
Additional file 2).
In all three species, glutaraldehyde or FAA fixation
resulted in the most tissue shrinkage, and for A. thaliana
and barley, 70% ethanol fixation gave similarly poor results. The procedure which consistently resulted in less
than 8% shrinkage for all three species was fixation in
methanol, followed by transfer to ethanol for 1 h then
CPD (Figure 1). A visual comparison of A. thaliana leaves
prepared by glutaraldehyde fixation and methanol fixationethanol dehydration shows the reduction in leaf area after
glutaraldehyde fixation and CPD compared to fresh tissue
(Figure 2).
Note that absolute ethanol, acetone and methanol rapidly penetrated tissues, causing both A. thaliana and
barley leaf tissue to sink immediately without vacuum
treatment. In comparison, tissues sank after light vacuum treatment in 70% ethanol, or only after extensive
vacuum treatment in glutaraldehyde and FAA fixatives.
An advantage of the methanol-ethanol treatment is that
tissue can be left uncut since solvent penetration is very
quick; for example, A. thaliana leaves were only cut at
the petiole, while barley and cotton leaves were cut on
four sides to fit the critical point drying basket.
Effect of fixation methods on epidermal cell morphology
Effects on tissue morphology generally reflected effects
on tissue dimensions, as seen in Figure 3, in which
methods causing the most to least changes to surface
morphology are presented sequentially from Figure 3A-F.
Using A. thaliana as an example since this tissue was
more sensitive to the different fixation procedures than
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%Loss or gain of tissue area
35
30
25
Arabidopsis
20
Cotton
Barley
15
10
5
0
FAA
Glut
70 eth
Meth
Meth-eth
100eth
Acet
Figure 1 Effect of SEM processing on dimensions of A. thaliana, cotton and barley leaf tissue. Loss or gain in tissue area after processing
through to critical point drying is expressed as percentage of original (fresh) tissue area. Treatments: FAA = FAA fixation; Glut = 3% glutaraldehyde;
70 eth = 70% ethanol fixation; Meth = Methanol fixation and dehydration followed by critical point drying in methanol; Meth-eth = methanol fixation
followed by ethanol dehydration; 100 eth = absolute ethanol fixation; Acet = acetone fixation followed by ethanol dehydration (details in text).
Bars are standard errors, 10 replicate tissue pieces were processed for glutaraldehyde, FAA, 70% ethanol and the 2 methanol treatments, and 16-20
replicates were processed for the absolute ethanol and acetone treatments. Only acetone treatment of A. thaliana leaf pieces resulted in overall tissue
swelling (i.e., an average positive value), indicated by a dark grey-filled bar. Arrows indicate treatments in which some replicates swelled and others
shrank, giving large standard errors.
Figure 2 Effect of SEM processing on A. thaliana leaf dimensions. Visual comparison of glutaraldehyde and methanol-ethanol (C,D) fixation
procedures, showing before (A,C) and after (B,D) critical point drying. Some leaves were slightly curled at the margins (A); this curling did not
seem to change after processing (B), which was the same for curled leaves processed through all other fixative regimes. All images are at the
same magnification. Scale bar = 2 mm, shown in D.
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Figure 3 Effect of SEM processing on morphology of A. thaliana leaf epidermal cells. A, FAA fixation; B; Glutaraldehyde fixation; C, 70%
ethanol fixation; D, methanol fixation followed by critical point drying in methanol; E, 100% ethanol fixation; and F, methanol fixation followed
by ethanol dehydration and critical point drying in ethanol (details in text). Acetone fixation followed by ethanol dehydration showed similar
preservation of morphology to 100% ethanol fixation. Stars indicate partial cell collapse, black arrowheads show cell wall folding and white
arrowheads indicate cell wall wrinkles. s = stomata. All images are at the same magnification. Scale bar = 20 μm, shown in F.
cotton or barley, we saw that the most damaging method
was FAA fixation, which resulted in partial cell collapse,
folding and wrinkling of walls (Figure 3A). Stomatal pores
were also closed and wrinkled. There was a spectrum of
similar artefacts following the other fixation procedures,
however, solvent fixed-tissues (Figure 3C-F) appeared to
fare better than those fixed in FAA (Figure 3A) or glutaraldehyde (Figure 3B). Of the solvent-based procedures,
methanol fixation followed by ethanol dehydration then
CPD in ethanol (Figure 3F), resulted in the least cell wall
wrinkling, with negligible cell collapse or wall folding. The
effects of the different treatments on morphology of barley and cotton leaves (Additional file 3), were similar to
those observed in A. thaliana leaf surfaces, but were generally less marked.
The tissues analysed here were all of similar area, although barley and cotton leaves were cut before processing, whereas A. thaliana leaves were processed whole.
We examined the effect of tissue size by processing larger cotton leaf pieces using the methanol-ethanol fixation method outlined above, but they collapsed and
were destroyed in the CPD vessel (Figure 4A). These larger leaf pieces remained intact if left to dehydrate in
100% dry ethanol overnight (Figure 4B), indicating that
larger tissues can be processed using this protocol with
some modifications to ensure more complete dehydration before CPD.
Discussion
Our results reinforce previous observations that optimal
SEM processing protocols differ for different tissues.
Nevertheless, we found that of all the protocols tested,
simultaneous fixation and dehydration in methanol
followed by further ethanol dehydration (modified from
[10]) gave the most consistent preservation of tissue dimensions and surface morphology of critical point dried
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Figure 4 Effect of dehydration time on cotton leaf pieces. Pieces were fixed in methanol and dehydrated in ethanol for either 1 h (A) or
overnight (B) before CPD. Images are at the same magnification. Scale bar = 2 mm, shown in B.
plant tissues. Furthermore, the methanol-ethanol protocol is relatively non-toxic, quick and simple, and requires standard SEM preparation equipment.
We speculate that the differences in response of different tissues might arise from differences in leaf structure,
epidermal surface coatings (wax and silica deposits) or
wall composition between the three species. In addition,
dimensional changes during specimen preparation are
likely to be unequal in different parts of the tissue [7].
Since barley leaf epidermal cells are ordered in rows, this
tissue might resist shrinkage in one direction, whereas
there is no such directional organization of cells in cotton or A. thaliana leaves.
Methanol is a highly polar solvent similar in structure
to water; it rapidly penetrates tissues and might simply
replace water throughout the tissues. Even the walls of
plant cells comprise up to 70% water by volume [17,18].
Hence, these properties of methanol make it an ideal
fixative for SEM, since removal of water is critical for
good preservation. Further dehydration with ethanol appears to be important, perhaps to completely remove
water or to serve as a better intermediate solvent for
CPD. The other solvents used as primary fixatives (ethanol and acetone) caused highly variable tissue shrinkage
or swelling and their use is not recommended, unless
thoroughly tested for a given tissue.
Glutaraldehyde has proven to be a good fixative for
structural preservation for light microscopy and TEM,
but as shown here and by others [10], is not necessarily
a good fixative for SEM preparation. Overall, glutaraldehyde and FAA resulted in relatively poor preservation
of tissue dimensions and morphology, which might
reflect the different action of these fixatives and longer processing times required for these methods. Although these methods could be modified for different
tissues to improve preservation, the methanol-ethanol
protocol was more consistent and resulted in better
morphological preservation.
A general protocol for methanol-ethanol fixation for
SEM is outlined below:
1. Fix tissue in 100% methanol for 10 min or longer.
Vacuum infiltrate if the tissue does not sink.
2. Transfer to 100% dry ethanol (30 min).
3. Dehydrate further in 100% dry ethanol for
30 min (small tissues) or overnight (large tissues),
with further changes into fresh 100% ethanol if
required.
4. Critical point dry following the manufacturer’s
recommendations.
5. Mount tissue on SEM stub, coat with conducting
metal if necessary (depending on type of imaging
required e.g., no coating, carbon, or gold coating for
Variable-Pressure SEM or gold for conventional
high-vacuum SEM) and observe.
6. Before and after SEM observation, store in lowhumidity environment, e.g. desiccator or controlled
humidity cabinet.
Conclusions
We found methanol to be a superior fixative and initial
dehydrating agent for SEM processing of plant tissues.
As noted earlier [10] and expanded on here, methanol
fixation and dehydration followed by further ethanol dehydration resulted in better preservation of surface
morphology and most consistent preservation of tissue
dimensions compared to other solvent-based and aqueous fixation procedures. As with all procedures, optimal
fixation and dehydration times should be empirically determined for a particular tissue type and size.
Talbot and White Plant Methods 2013, 9:36
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Methods
General methods
Relatively flat leaves from 2-3 week old agar-grown
Arabidopsis thaliana (L.) Heynh (Columbia) seedlings
were cut at the petiole. Leaves were harvested from
young barley (Hordeum vulgare L., Golden promise variety) or cotton (Gossypium hirsutum L.; Coker variety)
plants and 2-3 mm pieces were cut from the middle of the
leaf with a sharp double-edged razor blade. Leaves or leaf
pieces were imaged on a Leica MZFLIII dissecting microscope within 1 min, then transferred to fixative solution.
Ten to 20 replicate samples were processed for each fixative regime. After fixation and dehydration steps (different
methods outlined below), tissue was critical point dried
in an Autosamdri-815 automatic critical point drier
(Tousimis Research Corporation, Rockville USA) with
a 20 min purge time. Tissue was imaged again under
the dissecting microscope within an hour of completion
of CPD. Images of fresh and post-critical point dried tissue
were converted to greyscale and thresholded in Fiji (Image
J version 1.47 h; [19]) for analysis of tissue area. To determine how the different preparation methods affected
tissue morphology, 6 replicate critical point dried leaves or
leaf pieces were mounted on aluminium stubs with doublesided sticky carbon tabs (ProSciTech, Qld, Australia),
coated with gold (~20 nm) using an Emitech K500X sputter coater (Quorum Emitech, Kent, UK) and imaged in a
Zeiss EVO LS 15 Scanning Electron Microscope at 15 kV
accelerating voltage. Two images were taken from each
replicate.
Fixation and dehydration protocols
FAA
Leaf tissue was fixed according to Bomblies et al. (2008)
[20]. Tissue pieces were immersed in FAA fixative
(3.7% v/v formaldehyde, 50% ethanol, 5% acetic acid)
and subjected to a light vacuum until the tissue pieces
sank. Tissue was then fixed overnight (approximately
18 h) at 4°C. Tissue was rinsed 3 times in 25 mM sodium phosphate buffer (pH 7) before dehydrating in an
ethanol series (30%, 50%, 70%, 95% and 100% dry,
30 min each step). 100% dry ethanol was changed twice
and the tissue was stored overnight at 4°C before CPD
the next day. Note that this protocol calls for an osmium
tetroxide post-fixation step before dehydration, which
reduces tissue charging when observed in the SEM.
However, we found that this step was generally unnecessary if the tissue was gold-coated before SEM imaging,
so the osmium post-fixation step was omitted.
Page 6 of 7
100 to improve penetration of fixative. For the first 1020 min of fixation, tissue was vacuum infiltrated with
fixative plus detergent until the tissue sank. After the tissue had sunk this was replaced with fixative minus detergent and left overnight at 4°C. The tissue was then
washed 3 × 10 min in 25 mM sodium phosphate buffer,
rinsed in distilled water and dehydrated through an
ethanol series in 10% increments, starting at 10%,
30 min each step. Once in 100% dry ethanol, this was
changed twice, 30 min each, then the tissue was left
overnight in 100% dry ethanol at 4°C before CPD the
following day.
70% ethanol
Leaf tissue was immersed in 70% ethanol for 1 h, with
vacuum infiltration within the first 5 min or until the tissue sank, if necessary, then dehydrated to 100% dry
ethanol in 10% steps, 30 min each step. The 100% dry
ethanol was changed twice, tissue was left overnight in
100% dry ethanol at 4°C then critical point dried the
next day.
Methanol
This method is based on Neinhuis and Edelmann (1996)
[10]. Leaf tissue was immersed in 100% dry methanol for
10 min, followed by 2 × 30 min changes in 100% dry
methanol; vacuum infiltration was not necessary as tissue sank immediately. Tissue was then critical point
dried immediately with methanol as the transitional
fluid.
Methanol-ethanol
This method is based on Neinhuis and Edelmann (1996)
[10]. Leaf tissue was immersed in 100% dry methanol for
10 min, followed by 2 × 30 min changes in 100% dry
ethanol (vacuum infiltration was not needed). Tissue
was then critical point dried immediately with ethanol as
the transitional fluid. To explore this method further we
fixed larger cotton leaf pieces as described above. One
lot of tissue was then critical point dried immediately as
above, another lot was transferred into fresh dry ethanol
and left overnight at 4°C before critical point drying the
next day.
100% ethanol
Leaf tissue was immersed in dry 100% ethanol for
10 min, followed by 2 × 30 min changes in 100% dry
ethanol (vacuum infiltration was not needed). The tissue
was then critical point dried in 100% dry ethanol.
Glutaraldehyde
Acetone
Leaf tissue was immersed in standard fixative for SEM
(e.g., [3,8]) consisting of 3% glutaraldehyde in 25 mM sodium phosphate buffer (pH 7) and with 0.01% Triton X-
Leaf tissue was immersed in dry acetone for 10 min,
followed by 2 × 30 min changes in 100% dry ethanol (vacuum infiltration was not needed). The tissue was then
Talbot and White Plant Methods 2013, 9:36
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critical point dried in 100% dry ethanol. The tissue was
transferred to ethanol since acetone is not recommended
for use in the Autosamdri-815 critical point dryer.
Additional files
Additional file 1: Effect of absolute ethanol fixation and
dehydration and critical point drying on A. thaliana leaf area; raw
data used for graph in Figure 1. ‘Fresh’ refers to area (mm2) of fresh
tissue, while ‘CPD’ refers to area of tissue after critical point drying. Also
shown is the % loss (indicated by negative values) or gain in area after
processing.
Additional file 2: Effect of 100% acetone fixation, ethanol
dehydration, and critical point drying in ethanol, on A. thaliana leaf
area; raw data used for graph in Figure 1. ‘Fresh’ refers to area (mm2)
of fresh tissue, while ‘CPD’ refers to area of tissue after critical point
drying. Also shown is the % loss (indicated by negative values) or gain in
area after processing.
Additional file 3: Effect of SEM processing on morphology of
barley (A, B) and cotton (C, D) leaf epidermal cells, processed by
FAA (A, C) or methanol-ethanol (B, D) fixation. Stars indicate partial
cell collapse, white arrowheads indicate cell wall wrinkles. s = stomata.
All images are at the same magnification. Scale bar = 30 μm, shown in D.
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
MT contributed to conception and design of the study, carried out all
experiments and analysis and drafted the manuscript. RW contributed to
conception and design of the study and drafted the manuscript. Both
authors read and approved the final manuscript.
Page 7 of 7
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doi:10.1186/1746-4811-9-36
Cite this article as: Talbot and White: Methanol fixation of plant tissue
for Scanning Electron Microscopy improves preservation of tissue
morphology and dimensions. Plant Methods 2013 9:36.
Acknowledgements
We thank Tobias Baskin and two anonymous reviewers for helpful
comments on the manuscript.
Received: 2 July 2013 Accepted: 30 September 2013
Published: 2 October 2013
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